Field of the Invention
This invention relates to ambient-temperature stable compositions containing biopharmaceuticals and methods for formulation thereof; and more particularly, to such ambient-temperature stable compositions containing inactivated but therapeutically active (IBTA) microorganisms, proteins, antigens, or a combination thereof.
Description of the Related Art
Definitions
For purposes of this document, the term “microorganisms” is intended to include: viruses; bacteria; archaea; fungi; protista, yeast, and the like.
The terms “vaccine”, “biopharmaceutical”, “biotherapeutic”, “probiotic” and “biologic”, are each interchangeable herein, and each is intended to be defined as a composition containing at least a portion or component of a biological source. Examples of biopharmaceuticals, for purposes of this document, include, but are not limited to: microorganisms, proteins, and antigens.
In addition, “biopharmaceuticals”, “therapeutics”, “vaccines” and “probiotics” are each intended to be interchangeable herein, and collectively may be generally defined as “biopharmaceuticals”.
The term “inactivated but therapeutically active (IBTA) biopharmaceutical is interchangeable with the term “inactivated but potent vaccine” and “inactivated but immunologically active vaccines”.
The term “vaccine potency” as used herein, includes: survival rate of microorganisms, therapeutic activity of biopharmaceuticals, vaccine immunological activity, enzymatic or other activity of proteins cytokines and other macromolecules. Survival yield of viruses and bacteria and other microorganism means the same and will be used to address a measure of specific positive quality of the biologicals to which the method of this invention will be applied or used.
The term “thermos-stability” is defined as stability at all ambient temperatures from −20° C. to +37° C., for at least a year, with loss in the potency during 1 year storage below 0.5 logs even at +37° C.
The terms “therapeutically active” and “immunologically active” are each interchangeable herein, and each is intended to be defined by the common definition in the art.
The terms “sterilized” and “inactivated” are each interchangeable herein, and each is intended to be defined by the common definition in the art.
The terms “preserved”, “stabilized”, and “immobilized” are each interchangeable herein, and each is intended to be defined by the common definition in the art.
The term carbohydrate glass includes: any glass comprising a carbohydrate, but may further comprise any of: buffers, salts, amino acids, polymeric protectants, or a combination thereof.
As used herein “ambient-temperature stable” when used in conjunction with “biopharmaceutical”, “biologic”, or “biotherapeutic” is defined as a composition containing at least a portion or component of a biological source that is immobilized in amorphous carbohydrate glass at ambient temperature (from −20° C. to 37° C.) to ensure ambient-temperature stability. The biopharmaceutical immobilized in carbohydrate glass is generally dried at temperatures above ambient, often by way of several drying steps, in order to yield sufficient stability at ambient temperature.
Finally, for purposes herein, the terms “immobilized”, “preserved” and “stabilized” are used interchangeably, depending on whether the associated biopharmaceutical is a virus, bacteria, etc., and are intended to be defined by the common definition in the art.
Vaporization comprises three different processes, including: boiling, sublimation, and evaporation. Where sublimation is transformation from a crystallized into vapor gas phase. Boiling is a result of formation of vapor bubbles in the body of the superheated liquid. Superheating is necessary for the bubble nucleation for boiling to happen. Evaporating, also known as desorption, is transformation from liquid into vapor gas phase during which no boiling occurs and water molecules leave an aqueous liquid from its surface. Evaporation does not require an overheating.
Now describing the use of certain biopharmaceuticals as concerned herein within the context of their use as a therapeutic vaccine or other treatment platform.
Live Attenuated Vaccines (Virus and Bacteria)
It is well known that there can be a significant risk in administering live vaccines and other biopharmaceuticals containing microorganisms, even when attenuated.
Many conventional viral and bacterial vaccines are presently administered in a live attenuated state. These include attenuated pathogens used to treat the specific disease that they cause, and vectors for delivery of agents for gene therapy. A known risk of producing a live attenuated vaccine as opposed to an inactivated vaccine is the risk of reversion for virulence in the host. There is also a possibility for reaction to the production or attenuation process, such as, for example, egg allergy. Each vaccine released to the market for delivery in this state has been approved through clinical human trials to have a certain degree of safety in the general population, but there are often small subpopulations that are at a higher risk of negative response to the treatment. Immunocompromised individuals are at obvious risk for this response as they are often unable to fight off even slightly virulent biologicals. However, people are not always aware that they may have a weakened immune system, leaving a possibility of danger even in the typically-healthy patient. There are other factors such as genetic susceptibility that may leave certain groups of people more vulnerable to adverse vaccination responses. Effects can range from redness and swelling to mortality in some cases.
Probiotics
Other organisms with potential risk in certain populations include live probiotic bacteria. Historically defined by the World Health Organization (WHO) as “live microorganisms that, when administered in adequate amounts, confer a healthy benefit to the host”; these bacteria are meant to supplement the innate healthy flora and help to protect against pathogens. Protection occurs through various mechanisms. By ‘crosstalk’ between the bacteria and epithelial cells mediated by toll like receptors and small molecules, they are able to modulate both innate and adaptive mucosal immunity. Their adherent properties help to heal and maintain the tissues lining the digestive tract, sealing the tight junctions between the cells to reduce permeability. The typical dose of probiotic supplements number in the billions, but is only a small contribution to the trillions of bacterial cells in the human body. Large numbers of healthy bacteria protect against proliferation of pathogens in the body by competitive exclusion and by physical cell binding to facilitate pathogen elimination from the system. Prominent concerns with probiotics are in individuals with gastrointestinal disorders, chronic illness, immunocompromise, or in premature infants. Documented adverse reactions include sepsis, nonspecific or aggressive immune response, and endocarditis among others. Other potential side-effects are bacterial acquisition of virulence factors or resistance genes, the spread of those acquired factors to other intestinal bacterial populations, or translocation to blood or other tissues causing bacteremia.
Inactivated Vaccines
“Inactivated vaccines” are the most common example of killed therapeutically active microorganisms. Some of the most notable inactivated viral vaccines include Inactivated Poliovirus (IPV), Hepatitis A, inactivated rabies, and injectable seasonal influenza vaccines. Inactivated bacterial vaccines include those against typhoid, cholera, plague and pertussis. Vaccines of this type are typically created by subjecting cultured organisms to high heat, radiation, or chemicals such as formaldehyde or formalin. Because the replication components are destroyed there is no risk of genetic reversion to virulence as with live vaccines. However inactivation processes can be harsh on the organisms and inefficient, often destroying a large proportion of the immune-stimulating components. For example heat and chemical inactivation methods destroy not only the nucleic acids and ability of microorganisms to replicate, but also many epitopes, capsid proteins, intracellular and membrane associated proteins and other molecules relatively intact and recognizable by the immune system or responsible for other therapeutic activity. Inactivated vaccines appear to be more stable than live vaccines because the most fragile components that would normally be lost with time and temperature have already been inactivated, or because it is difficult to accurately evaluate losses biopharmaceutical potency or therapeutic functions in inactivated specimens. One drawback of non-replicating organisms is that they are less potent and have a shorter period of protection in the system, often requiring boosters to maintain immunity long-term. Another is that a larger dosage must be given to counter the proportion of destroyed organisms and the fact that they will not replicate to higher titers in the host.
In addition, development of ambient-temperature stable inactivated vaccines and other biotherapeutics is a challenge because in order to demonstrate stability of inactivated vaccines after preservation, animal immunogenicity studies (or therapeutic potency studies) must be run at each evaluation point. In this the embodiments disclosed herein, we suggest thermostabilizing (at ambient and higher temperatures) the biotherapeutic component first, and subsequently inactivating such by irradiation. This will allow testing and verifying survival (or activity) of preserved specimens over time before inactivation, without a need to conduct animal studies at each stability time point.
Killed Probiotics
‘Killed probiotics’ are increasing in popularity in various industries. Although they are typically administered alive so they have the ability to colonize the gut for prophylactic protection, an increasing number of studies have shown that various strains have a therapeutic effect when administered in an inactivated form. The mechanism of therapeutic action in this case is not yet fully elucidated. This approach to probiotic use has shown potential for use in the agriculture and livestock industry where widespread antibiotic use is often standard to stave off diseases prevalent in the overcrowded and unsanitary conditions. Protein components of the probiotic surface strongly aggregate to each other and adhere to the mucin and extracellular matrix materials of cells. When adhered to the mucin membrane of the intestine they create a barrier to block the pathogenic cells from attaching. They also function to adhere to the pathogens themselves, coating their attachment structures and flushing them out of the system.
Killed probiotic therapy uses a very large dose of bacteria to compensate for the fact that they will not replicate in the host, and the methods of killing are damaging to the cells. Most studies use harsh heat or chemical treatment to kill the cells which can be detrimental to the external proteins and epitopes which are used for adhesion and cellular recognition, therefore leaving a less potent therapy.
Therefore there is an urgent need in developing more gentle methods for inactivation of biotherapeutics and production of ambient-temperature stable inactivated vaccines and other biotherapeutics.
After many methods of production or purification of active biologicals including cytokines, toxins, therapeutic proteins or protein antigens from blood, cell cultures, milk, plant extracts or other biological liquids, the products could be contaminated with live microorganisms. We found that inactivation of contaminating microorganisms could be performed by irradiation of the active biologicals immobilized in carbohydrate glasses at AT without destroying their therapeutic activity (i.e. immunological activity of epitopes)
Stabilization Technologies
Currently, it is well recognized that the long-term stabilization of biologicals requires arresting molecular mobility to stop the degradation processes during storage. This can be achieved only by vitrification, which is the transformation from a liquid into a supercooled or supersaturated, noncrystalline, amorphous solid state, known as the “glass state”. The basic premise is that the high viscosity of the glass state will arrest all diffusion-limited physical processes and chemical reactions, including the processes responsible for the degradation of biological materials. This premise is based on Einstein's theory that establishes the inverse proportionality between viscosity and molecular mobility (or diffusion coefficients of molecules). In general terms, glasses are thermodynamically unstable, amorphous materials; however, they can maintain the same state for long periods of time because of their very high viscosity (1012-1014 Pa*s); for example, a typical liquid has a flow rate of 10 m/s compared to 10−14 m/s in the glass state.
Depending on the composition, a biological suspension could be transferred into the vitrified state, by cooling, increasing in hydrostatic pressure, or a combination thereof, at different temperatures, if the cooling rate is sufficiently high to avoid formation of the crystalline phase. For example, pure water could be vitrified by cooling below −148° C. Preservation of cells and other biologicals by cryo-vitrification has been introduced as an alternative to preservation by freezing to avoid freeze-induced damage of biologicals. To achieve cryo-vitrification before cooling cells are typically equilibrated in concentrated solutions of low toxicity polyols (protectors) like DMSO, glycerol, Ethylene Glycol, etc. These solutions help to avoid formation of ice crystals (freezing) but have very low glass transition temperatures (Tg), i.e., below −100° C., because of high water concentration and low Tg of pure protectors. Therefore for preservation of biologicals above −100° C. one should use protectors with higher Tg.
In general the presence of water in a sample has a strong plasticizing effect, which decreases the glass transition temperature (Tg) and thus limits stability at higher temperatures (AT). For example, for water, Tg is about −148° C., for 80% sucrose, Tg is about −40° C.; Tg of 99% sucrose is about +52° C. Therefore, if biologicals are to be preserved without degradation at an ambient temperature, they must be strongly dehydrated before transformed in the glass state by cooling. Similar to that for cryo-vitrification in the dry immobilized state, biopharmaceuticals are dormant, but can be returned to the active (or live) state after reconstitution with water.
Dehydration (drying) can be very damaging to vaccines and other fragile biologicals if performed in the absence of protective molecules (i.e. sucrose, trehalose, etc.) that adsorb at the surface of biological membranes and macromolecules and replace water of hydration at the surfaces, and this way protects the biologicals from destruction associated with hydration forces that arise during dehydration. Because of this, proper selection of the protective molecule is a key to a successful stabilization of biologicals at ambient temperatures without loss of their activity.
Evaporative Drying (Desorption)
A simple method of drying that can be applied for long-term stabilization of biologicals at ambient temperatures is an evaporative drying. During evaporation, water leaves a specimen from its surface into a dry air or vacuum. However, before reaching the surface water should diffuse through the body of the specimen. Thus, evaporative drying is a diffusion-limited process. Because of this, desorption could be applied only for drying of small drops or very thin specimens with large surface to volume ratios. After desorption, a specimen should be cooled to achieve the glass state.
Evaporative drying (ED) was very successfully applied for producing AT stable formulation of many biopharmaceuticals including vaccines. However, (ED) is very difficult to scale for most applications. Because of this, freeze-drying (FD) and spray-drying (SD) technologies are conventionally used as the primary methods for the stabilization of vaccines and fragile pharmaceuticals in the dry state. However, there were fundamental reasons preventing greeze-drying and spray-drying from delivering ambient-temperature stable vaccines and many other biologicals.
Freeze-Drying (Lyophilization)
Freeze-drying has been unsuccessful in delivering ambient-temperature stable vaccines.
Despite its limitations and shortcomings, freeze-drying has remained, for more than 50 years, the primary method to stabilize fragile biopharmaceuticals and biologics (vaccines, therapeutic proteins, probiotics, etc.) in the dry state. This is, in part, because of erroneous conventional belief that drying at low temperature would be less damaging, and in part because, during many years, there had been no alternative scalable drying technologies available. Conventional freeze-drying requires a very long time, excessive costs, and in many cases, produces low yields because it is a very damaging process for many biopharmaceuticals. Freeze-dried biopharmaceuticals, such as vaccines, require refrigeration and a cold chain to maintain stability and viability during transportation, storage, and delivery to the point of use. Lyophilization-induced injury happens both during freezing and during subsequent ice sublimation from frozen specimens at intermediate low temperatures (between −50° C. and −20° C.). It is at these temperatures that most damaging cryochemical reactions occur.
Spray Drying
Spray drying has also been unsuccessful in delivering ambient-temperature stable vaccines.
Spray drying is a scalable process for drying of biological specimens sprayed in a dry environment at high temperatures. Conventionally spray drying was used as a sterilization process for milk and other biological liquids during drying. Removal of the water from the small drops (microsphere) during spray drying occurs by evaporation, which is limited by diffusion of the water from the middle of a microsphere to its surface. Characteristic time (t) of the diffusion relaxation in the drop with diameter (d) is about t=d2/D, where D is the water diffusion coefficient. In water, D=10−5 sm2/sec and for small drops with diameter d=10μ and t=0.1 sec. However, for drops containing concentrated solutions (syrups), it will greatly increase because D of syrups is smaller than 10−5 sm2/sec by many orders of magnitude. The solution to slowing the drying with water leaving the drop is increasing the drying temperature, which could damage the vaccine. Here it is important to note that when D becomes very small it will take many hours to remove water even from a micron size particles. This is a major reason explaining why it is very difficult to reach high glass transition temperature after spray-drying without overheating and destroying the vaccine activity.
Vacuum Foam Drying
Vacuum Foam Drying was introduced to scale up desorption. In brief, the technique of foam drying is composed of boiling a very concentrated and viscous aqueous solution (Syrup) under vacuum at ambient temperature (AT), such that it transforms into foam. During this process shear stresses that occur in the viscous liquid during the growth of vapor bubbles nucleate new bubbles that split thick films into thin films that can quickly dry under vacuum. The large surface area of the foam allows efficient desorption of the water from the bubbling syrup and solidification of the material in the foam; and at the end of the vacuum drying period the solution becomes a mechanically stable dehydrated foam.
Preservation by Vaporization (PBV)
Preservation by Vaporization (PBV) is a core technology for development and production of ambient-temperature stable probiotics, live attenuated vaccines (LAV) and other biopharmaceuticals, and is described in detail in commonly owned WO 2005/117962. PBV is a scalable, reproducible, and automatable state of the art generation of vacuum foam drying that is free from the drawbacks of Preservation by Foam Formation (PFF).
PFF is described in “Preservation by Foam Formation”, U.S. Pat. No. 5,766,520 (1998). Additionally, PFF is described in Bronshtein, V. article “Preservation by Foam Formulation, an Alternative to Freeze-Drying” (Pharmaceutical Technology. 28: 88-91, 2004); which describes an alternative to freeze-drying for production of ambient temperature stable vaccines.
PBV can be performed in unit dose format (in vials) and/or in bulk format (in trays, bags, or other containers) using conventional freeze-drying equipment. In addition, PBV could be executed as a barrier continuous load process that is ideal for the production of biopharmaceuticals.
PBV overcomes deficiencies of conventional scalable drying technologies such as freeze-drying and spray-drying. Today foam drying is the only scalable drying technology that has been proven to deliver fragile ambient-temperature stable biologicals like live attenuated vaccines.
Ambient-temperature stable vaccines and other biologicals formed by PBV have a shelf life at ambient temperatures measured in years and are suitable for needle-free delivery using dry powder inhalers, tablets, dissolvable films, microneedle patches, suppositories, ointments, creams, and enteric coated capsules among others, including the embodiments as disclosed herein.
It has been recognized that there is a need for mucosal and transdermal delivery of biopharmaceuticals, especially in regions of the World where professional medical care is limited, for example, in developing countries. With this in mind, there is a significant need for delivery platforms, such that certain biopharmaceuticals can be manufactured, stored, transported, and ultimately administered to a patient, ideally without a syringe delivery platform. Thus, modern solutions containing improvements in the art are necessary to meet these current demands.